Active dye and reaction conditions
Check moisture exposure, delays after dye preparation and the actual reaction pH. NHS esters hydrolyze in water, and increasing pH also accelerates hydrolysis. Address competition from Tris, glycine or carrier proteins before comparing dye input.
Use measurements to locate the problem
Measure DOL and target concentration after purification. Low DOL points to active-dye availability or accessible amines; low recovery calls for checks of precipitation and purification losses. If DOL rises while specific binding falls, compare lower dye inputs. Match target concentration and instrument settings across groups.
Choose the next check from the result
Compare at matched target concentration and acquisition settings before attributing a weak signal to reaction failure.
| Observation | First check |
|---|---|
| Low DOL, normal recovery | Moisture or hydrolysis, competing amines, actual pH, accessible primary amines |
| Adequate DOL, low target recovery | Precipitation, adsorption to columns or membranes, collected volume |
| High DOL, reduced binding | Compare unlabeled and preparation-only controls, then test a lower dye input |
| Bright negative sample | Residual dye, working concentration, nonspecific binding and channel spillover |